Snake hemoglobins can be used as models to understand the transition by mammalian hemoglobins from their deoxygenated form to their oxygenated form. The advantage of snake hemoglobins over mutant or chemically engineered mammalian hemoglobins is that the former naturally presents many substitutions of important amino acids at the packing (a,01) and sliding (a,01) interfaces which endow them with special allosteric properties and a unique oxygen-transporting mechanism. They also present four different chains (A, B, C, and D) as opposed to only two distinct mammalian chains, a and P. The present study was conducted on Liophis milaris hemoglobin, a Brazalian semi-aquatic snake. The four hemoglobin chains, A(pl) ), B (aA), C(aD ) and D(pA), were separated, identified and characterized by RP-HPLC, amino acid analysis, Edman sequencing and enzymatic digestions by endo and exopeptidases. Sequencing results provided complete information for chains A(P), C(a) and D(P). Even though chain B((x) presented heterogeneous forms (which could not be separated by the analytical methods mentioned), sequence information was obtained for the mixture of its variants. The characterization of these variants, as well as confirmation of the sequence data, was obtained only after extensive mass spectrometric analysis.